Clear-cell renal cell carcinoma (ccRCC) is the most prevalent and malignant histological type of kidney cancer, for which there are no effective methods of treatment for metastatic disease. ccRCC is characterized by early loss of the von Hippel-Lindau tumor-suppressor gene (VHL) in a majority (60%-80%) of tumors. One major oncogenic effect of VHL loss is the induction of hypoxia-inducible factor (HIF) and HIF-regulated genes leading to angiogenesis, which supports tumor growth by providing nutrients from extracellular sources. In our previous work, we discovered that loss of VHL regulates pathways that secure nutrients from intracellular sources through autophagy: VHL, by inducing expression of miR-204, targets the autophagic regulator, LC3B, and inhibits oncogenic autophagy. By inhibiting HIF, VHL induces expression of LC3C, an LC3B ortholog, which has tumor suppressive activity. This proposal is focused on the TRPM3 channel (transient receptor potential M3 Ca2+-permeable, nonselective cation channel), which is the protein product of the host gene that encodes miR-204 in its intron 6. We discovered that TRPM3 plays a pro-oncogenic role in ccRCC. TRPM3 is overexpressed in ccRCC and regulates two autophagic pathways; it is necessary for LC3B/LC3A autophagy and inhibits LC3C-mediated autophagy. In contrast to miR-204, TRPM3 is repressed by VHL at the level of protein accumulation. Our hypothesis is that the effects of TRPM3 on autophagic programs play an essential role in its ability to promote RCC tumor growth, and that the effects of TRPM3 on autophagic programs are mediated through regulation of intracellular calcium. In Aim 1, we will investigate the molecular mechanism by which VHL represses TRPM3, with the leading hypothesis that VHL-induced miR-204 targets and represses expression of its host gene product. In Aim 2, by manipulating expression of different autophagic regulators affected by TRPM3, we will identify the autophagic pathways essential for the oncogenic activity of TRPM3. In Aim 3 we will manipulate extracellular and intracellular Ca2+ and Ca2+-regulated kinases implicated in the regulation of autophagy and determine the effects on autophagy in cells that are VHL(-) or VHL(-) with TRPM3 knocked down. In Aim 4 we will study the mechanisms of transcriptional induction of LC3C in cells with knockdown of TRPM3. With this proposal, we expect to further establish VHL as a major regulator of autophagy.